Ensembles of nanoelectrodes as electrochemical and electrochemiluminescence sensing platform for molecular diagnostics

Abstract

Nanoelectrode ensembles (NEES) are prepared, functionalized and tested to prepare enzymatic and affinity sensors suitable for advanced molecular diagnostics purposes, namely the development of a miniaturized glucose biosensor and the preparation of novel electrochemical (EC) and electrochemiluminescence (ECL) immunosensors for celiac disease diagnostics. For the first goal, a second generation enzymatic microbiosensor was developed exploiting the properties of NEEs prepared by electroless gold deposition in track-etched polycarbonate (PC) membrane. The micro-NEE glucose biosensor (overall radius of 400 µm) was obtained by immobilizing glucose oxidase (GOx) on the nonconductive PC component of the NEE, while the Au nanoelectrodes were used exclusively as transducers. The (Ferrocenylmethyl)trimethylammonium cation (FA+) was used as the redox mediator. The proposed biosensor showed outstanding analytical performances with a detection limit of 36µM for glucose. The second goal concerns with celiac disease (CD) diagnostics. CD is a chronic small intestinal autoimmune enteropathy triggered by exposure to dietary gluten in genetically predisposed individuals. The prevalence rate in the general population is about 1%; but the incidence rate is dramatically increasing over time. People with CD exhibit abnormally high blood levels of anti-tissue transglutaminase (anti-tTG) antibodies, suitable as biomarkers for its diagnosis. Existing serological diagnostic techniques lack the desired level of sensitivity and specificity so that a confirmatory biopsy test is required. To overcome this limitation, EC and ECL immunosensors are proposed and studied. The two kinds of immunosensors employ the same biorecognition platform, based on tTG as biorecognition element and NEEs as electrochemical transducers. The EC and ECL sensors differ in the type of label used to develop the detection signal, and the method of signal detection. By exploiting the high affinity of PC for proteins, the capture antigen tTG is at first immobilized on the PC of the NEEs obtaining tTG–NEEs, which can capture the anti-tTG antibodies. For EC detection, the bound anti-tTG is reacted with a secondary antibody labeled with horseradish peroxidase, so that signal is detected using H2O2 as enzyme substrate and hydroquinone as redox mediator to generate the detection signal. For ECL detection, following the capture of anti-tTG, the immunosenser is reacted with a biotinylated secondary antibody to which streptavidinated Ru(bpy)3+2 luminophore can be bound. In this design, the electrochemical reaction and the location of the immobilized biomolecules where ECL is emitted are spatially separated. Application of an oxidizing potential in tripropylamine (TPrA) solution generates an intense ECL, suitable for the sensitive detection of anti-TG, at a potential much lower than the ECL initiated by the electrochemical oxidation of 〖Ru(bpy)〗_3^(2+). Since the luminophore does not undergo direct electrooxidation at the surface of the nanoelectrodes, the ECL signal is generated only by the TPrA electrooxidation. Note that TPrA acts as redox mediator and ECL coreactant. Both EC and ECL sensors are applied to human serum samples, showing to be suitable to discriminate between healthy and celiac patients. A comparison between the two approaches indicates that the lowest detection limit, namely 0.5 ng mL-1 of anti-TG, is achieved with the ECL immunosensor.